This invention relates to arrays, particularly biopolymer arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include features (sometimes referenced as spots or regions) of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. The array is xe2x80x9caddressablexe2x80x9d in that different features have different predetermined locations (xe2x80x9caddressesxe2x80x9d) on a substrate carrying the array.
Biopolymer arrays can be fabricated using in situ synthesis methods or deposition of the previously obtained biopolymers. The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA). In situ methods also include photolithographic techniques such as described, for example, in WO 91/07087, WO 92/10587, WO 92/10588, and U.S. Pat. No. 5,143,854. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different feature locations on the substrate to yield the completed array. Washing or other additional steps may also be used. Procedures known in the art for deposition of polynucleotides, particularly DNA such as whole oligomers or cDNA, are described, for example, in U.S. Pat. No. 5,807,522 (touching drop dispensers to a substrate), and in PCT publications WO 95/25116 and WO 98/41531, and elsewhere (use of an ink jet type head to fire drops onto the substrate).
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require the manufacture and use of arrays with large numbers of very small, closely spaced features.
The arrays, when exposed to a sample, will exhibit a binding pattern. The array can be interrogated by observing this binding pattern by, for example, labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), scanning an interrogating light across the array and accurately observing the fluorescent signal from the different features of the array. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample. Peptide arrays can be used in a similar manner. Techniques for scanning arrays are described, for example, in U.S. Pat. No. 5,763,870 and U.S. Pat. No. 5,945,679. However, the signals detected from respective features emitted in response to the interrogating light, may be other than fluorescence from a fluorescent label. For example, the signals may be fluorescence polarization, reflectance, or scattering, as described in U.S. Pat. No. 5,721,435.
Array scanners typically use a laser as an interrogating light source, which is scanned over the array features. Particularly in array scanners used for DNA sequencing or gene expression studies, a detector (typically a fluorescence detector) with a very high light sensitivity is normally desirable to achieve maximum signal-to-noise in detecting hybridized molecules. At present, photomultiplier tubes (xe2x80x9cPMTsxe2x80x9d) are still the detector of choice although charge coupled devices (xe2x80x9cCCDsxe2x80x9d) can also be used. PMTs are typically used for temporally sequential scanning of array features, while CCDs permit scanning many features in parallel.
Laser output power in such array scanners may tend to drift over time. As described in U.S. Pat. No. 5,763,870, it is known to provide an integral power regulation sensor for a laser which is used to monitor laser output power. The power sensors (that is, laser light illuminance sensors) are connected to current-regulating circuitry that varies the supply current to the laser and responds to changes in output power. For gas lasers, the power sensors may be mounted internally and a beam splitter redirects a portion of the output beam energy to the power sensor, which may be a photodiode. For semiconductor lasers, the power sensor may be formed on the same substrate as the semiconductor layers that define the laser device, such as described in U.S. Pat. Nos. 5,323,026 and 4,577,320. However, the present invention recognizes that for some types of gas lasers it may be difficult to reliably switch between two power levels quickly, and that for diode lasers changing the laser power may have the undesired side effect of changing the wavelength.
The present invention further realizes that strong signals may occur in response to the interrogating light, either from bright features or from other components (for example, fluorescence of the glue holding an array substrate in a housing). In PMTs and other detectors (such as CCDs) very strong signals that are (depending on the type of detector) either spatially and/or temporally close to weak signals, may undesirably affect the latter. For example, a PMT reading a very bright signal at a given time, may change its sensitivity for a short time after this, or on a CCD detector very bright pixels may bleed their charge into adjacent ones. Extremely strong signals may even damage either kind of (normally expensive) detector. In any event, the accurate detection of signals from an array being interrogated by the scanner may be in doubt due to such effects.
The present invention realizes that it would be desirable then, to provide a technique for scanning an addressable array which allowed for rapid correction in variations in power of an interrogating light. The present invention further realizes that it would be desirable if some means were provided during array scanning, to limit exposure of a detector to very strong signals generated by features or other sites in response to the interrogating light. Additionally, the present invention also realizes that during the typically rapid scanning of an array, some types of light sources may not respond sufficiently rapidly to changes in power input (as discussed above) to allow for the corrections of the present invention while maintaining high array scanning speeds.
The present invention then, provides a method for use with an addressable array of multiple features of different moieties. These moieties may, for example, be polynucleotides (such as DNA or RNA) of different sequences for different features. In the method, an interrogating light is scanned across the array. This scanning can be accomplished, for example, by moving the interrogating light relative to the array, moving the array relative to the interrogating light, or both. The interrogating light is generated from a variable optical attenuator through which light from a light source has passed, and which optical attenuator is responsive to a control signal to alter the power of the interrogating light. Signals from respective features emitted in response to the interrogating light, are detected. A power of the interrogating light from the variable optical attenuator is also detected. An attenuator control signal is adjusted to alter interrogating light power, which adjustment is based on the detected power. The power may be detected and altered at any convenient times. For example, the power may be detected and altered during the scanning step (such as during a transition of the interrogating light from one row to another in the case where the array includes rows of features which are scanned row by row).
In a second aspect, the interrogating light is scanned across multiple sites on an array package which includes the array, and which scanned sites include the features. Signals emitted from respective scanned sites in response to the interrogating light, are detected. The interrogating light power is adjusted for a first site (which may or may not be an array feature) on the array package during the array scan based on some characteristic of the first site, such as location of the first site or a determination that the emitted signal from the first site will be outside a predetermined range (for example, a lower or upper limit) absent the altering. For example, the interrogating light power may be reduced based on a determination that the emitted signal from the first site will exceed a predetermined value, or based on location of the first site. If the determination is used this can be based, for example, on the results of a pre-scan or on the signal emitted from the first site when the interrogating light initially illuminates the first site. A pre-scan can include scanning an interrogating light across multiple sites on the array package using a lower sensitivity than that used in the previously mentioned scan (sometimes referenced as the xe2x80x9cmain scanxe2x80x9d). Lower sensitivity can be obtained by using a lower interrogating light power, or with an emitted signal detector of lowered light sensitivity. Signals emitted in response to this interrogating light are detected and the determination based on one or more of these signals obtained in the pre-scan. If interrogating light power is altered based on location of the first feature, this location can be based on a reading (machine or human) of an identification associated with the array package (such as an identification carried on the package).
In the second aspect, any alteration of the interrogating light power can be made using the results of a previously obtained calibration. In particular, the method may also include calibrating the interrogating light power versus a control signal, for a light system which provides the interrogating light of a power which varies in response to the control signal. Optionally, this calibration may be repeated before scanning and detection for each of multiple array packages. While various light systems may be used in the second aspect, a light system which includes a light source and an optical attenuator in the arrangement disclosed above, may for example be used.
The present invention further provides apparatus for executing methods of the present invention. In a first aspect, the apparatus includes the light source and variable optical attenuator, a scanning system to control scanning, an emitted signal detector, and a power detector to detect the power of the interrogating light. The apparatus also includes a system controller which receives input from, and controls the remainder of, the apparatus as required (including using location information or making determinations, as described above) such that the remainder of the apparatus can execute a method of the invention. For example, the system controller may adjust the optical attenuator control signal to alter interrogating light power, based on the power detected by the power detector. Such an apparatus can execute the first aspect of the method described above. In an alternative example, the system controller adjusts the interrogating light power for a first site on the array package during the array scan based on location of the first site or on a determination that the emitted signal from the first site will be outside a predetermined range absent alteration. Such an apparatus can execute the second aspect of the method described above. However, the system controller optionally, and preferably, receives input from, and controls the remainder of, the apparatus as required such that the same apparatus can execute both a method of the first and second aspects described above.
In one particular embodiment of the apparatus, the apparatus further includes a reader to machine read an identification carried on the array package. In this case, the system controller may determine the location of the first site based on the read identification. This determination can, for example, be made directly from data contained in the read identification or retrieved from a local or remote source using the identification.
The present invention further provides a computer program product for use in an apparatus of the present invention. Such a computer program product includes a computer readable storage medium having a computer program stored thereon which, when loaded into a computer of the apparatus, such as the controller, causes it to perform the steps required by the apparatus to execute a method of the present invention.
While the methods and apparatus have been described in connection with arrays of various moieties, such as polynucleotides or DNA, other moieties can include any chemical moieties such as biopolymers. Also, while the detected signals may particularly be fluorescent emissions in response to the interrogating light, other detected signals in response to the interrogating light can include polarization, reflectance, or scattering, signals. Also, the described methods in the present application can be used to alter the actual scanning pattern of the interrogating light (rather than, or in addition to altering the interrogating light power) on the array package, particularly based upon a read identification (such as a machine readable identification) carried on the array package. For example, the dimensions of the scanned area, and/or the number of scan lines and/or scan line spacing may be based on the read identification (with the required information being retrieved from the read ID or from a local or remote database).
The method, apparatus, and kits of the present invention can provide any one or more of the following or other benefits. For example, correction in the power of an interrogating light can be obtained. Also, exposure of a detector to very strong signals generated by features or other sites in response to the interrogating light, can be limited based on rapid altering of interrogating light power. This can avoid both detector damage and detector blinding (temporary, light-induced change of sensitivity) at high switching speeds with a simple apparatus. Further, alterations of interrogation light power may be obtained which may be faster than obtainable from altering power to some types of light sources, so that high array scanning rates can still be maintained.